Relevance of <scp>Howell‐Jolly</scp> body counts for measuring spleen function in sickle cell disease
Authors: Charlotte Pourdieu, Sara El Hoss, Enora Le Roux, Justine Pages, Bérengère Koehl, Florence Missud, Laurent Holvoet, Ghislaine Ithier, Malika Benkerrou, Zinedine Haouari, Lydie Da Costa, Wassim El Nemer, Sandrine Laurance, Yves Colin Aronovicz, Caroline Le Van Kim, Odile Fenneteau, Elodie Lainey, Valentine Brousse
Journal: American Journal of Hematology (2023)

The spleen is essential for clearing blood-borne infections because it both filters the blood and provides specific immune defense. The measurement of splenic function is, however, challenging in clinical practice.1 Historically, Howell–Jolly bodies (HJBs) or pitted cells have been used as surrogate markers of the spleen's functions, based on retrospective reports of elevated HJB counts in patients with severe and sometimes fatal invasive pneumococcal infections. More recently, automated counts of pitted cells or HJBs have enabled the spleen's function to be monitored and quantified more accurately.2 Susceptibility to pneumococcal infection is well-known in sickle cell anemia (SCA, i.e., Hb SS/Sβ°thalassemia disease) and has been attributed to early damage of the spleen. The implementation of anti-pneumococcal prophylaxis early in life has drastically decreased related mortality and morbidity rates in SCA children, but the probability of infection remains elevated throughout life and justifies the aggressive management of febrile episodes with intravenous antibiotics, particularly in younger children. In contrast, the few studies of susceptibility to pneumococcal infections in children with compound heterozygous hemoglobin SC (HbSC) disease have given conflicting results. In a recent report, the estimated incidence of invasive pneumococcal disease in children under 5 years of age in 2010–2018 (after the introduction of conjugated anti-pneumococcal vaccines) was 3.92/100 000 patient-years (PY) in children with SCA, 2.88/100 000 PY in children with HbSC, and 0.15/100 000 PY in the general population.3 In children with HbSC, a time lag in the onset of impaired splenic function in the second decade of life has also been suggested and the few reported fatal cases of invasive pneumococcal disease all occurred in children over the age of 9.4 Given the overall degree of uncertainty regarding the risk of pneumococcal disease in children with HbSC disease, the recommended prophylactic and therapeutic measures for pneumococcal infections in these patients vary markedly from one set of guidelines to another. In order to better characterize the infectious susceptibility of children with HbSC disease under the age of 10, we used a validated, high-throughput, minimally invasive flow cytometry HJB assay to quantify splenic function.5 For comparative purposes, we also studied children with SCA, controls, and splenectomized children. For the HbSC and SCA groups, we reviewed the children's medical files since birth for previous invasive infections, defined as an episode of sepsis with an identified bacterial pathogen (using cultures or PCR assays). Unselected consecutive patients with HbSC disease and SCA at steady state aged under 10 were enrolled by the sickle cell disease clinic team at Robert Debré University Hospital (Paris, France) between January 2021 and July 2021. All children were followed since new born screening at our institution. Children having received a blood transfusion in the previous 3 months were excluded from this study. The controls were children being followed for endocrine diseases, none of which involved an impairment in spleen function. Splenectomized children were selected consecutively from among the hospital's pediatric patients regardless of age given that over 3 months post-splenectomy, the rate of HJB remains stable.6 The study was approved by the local independent ethics committee (CEER, Hôpital Robert Debré, Paris, France; reference: 2020-527bis). The HJB count in whole blood samples was measured on at least 100 000 red blood cells (RBCs) using flow cytometry, as described previously.5 Samples were washed and stabilized in ID-CellStab (Bio-Rad, Hercules, CA) immediately after venipuncture and then analyzed within 48 hours of collection. Samples were analyzed on a flow cytometer (Navios, Beckman Coulter, Germany), using a FACSDiva software (version 8.0.1 BD Biosciences). The data were analyzed using the Flow Jo software (version 10). In an internal quality control check, we compared the FC-HJB data with manual HJB counts; as observed previously the two counts were linearly related.5 For descriptive and univariate analyses (based on the Mann–Whitney test), age was analyzed as a quantitative variable and as a categorical variable (<5 years and ≥5 years) using the 5-year threshold frequently applied in the literature on the risk of pneumococcal disease. A multivariate linear regression model was then used to study the association between the HJB count in the SCA group with hydroxyurea (HU) treatment (as a binary variable) and age (as a quantitative variable). In 2021, a total of 397 children with sickle cell disease <10 years were followed at our institution, with 79 children with HbSC <10 years, 318 with SCA (310 with HbSS and 8 with Sbeta° thalassemia). In this study, we enrolled 40 HbSC patients over a 6-month period, representing 51% of total HbSC patients. During the same period, 53 children with SCA disease, 22 controls, and 15 splenectomized patients were enrolled for comparison purposes. The overall median (range) age was 4.6 years (0.16–9.99). In inter-group comparisons (SCA vs. HbSC, SCA vs. controls, and HbSC vs. controls), the three groups did not differ significantly with regard to the median age. The spleen function assay (FC-HJB) showed that the median [IQR] HJB count in children with HbSC disease was very low (median: 16 [Q1:11.50–Q3:28.25]/100 000 RBCs) and was not different to that measured in controls (p = .96; Figure 1A). We did not observe an influence of age on spleen function when age was analyzed as a continuous or binary variable; p = .91. In contrast, the median HJB count was significantly higher in the SCA group than in the HbSC and control groups (p = .002 for children <5 and <.001 for children ≥5). The median HJB count in the SCA group remained significantly lower than the value observed in the splenectomized group (p < .0001). We noted great inter-individual variability in the HJB count in children with SCA (particularly those under 5), in line with the well-known phenotypic variability observed in SCA. The HJB count was found to increase with age in the SCA group (Pearson's correlation coefficient: 0.30, p = .03), indicating a progressive loss of splenic function over time, including in the second decade of life (Figure 1B). Regarding past history of invasive bacterial disease, one child with a normal HJB count in the HbSC group experienced a pneumococcal sepsis. Two children with SCA experienced a pneumococcal disease and they both had an elevated HJB count. In children with SCA, there was a statistically significant difference in the HJB count between those treated with HU and those not (p < .001, Mann–Whitney test). However, it is noteworthy that only 21 (39.6%) of the 53 children with SCA were treated with HU, and that most (71.4%) of the treated children were over the age of 5. We sought to take account of the treatment-age interaction in a linear regression model. The results showed a significant impact of age (p = .04 (0.002; 0.29); Beta = 0.15) and a non-significant impact of HU treatment (p = .08 (−0.10; 1.56); Beta = 0.73) indicating that age explained the HJB counts more fully than the treatment did and suggesting that splenic function falls over time, regardless of treatment. One of the key findings of the present study is that splenic function measured with a validated, high-throughput method is preserved during the first decade of life in children with HbSC. Our results confirm prior reports of low rates of HJBs in HbSC patients under 10 and argues for extendable results in other cohorts or settings.4 There is, however, a contradiction between normal spleen function (as measured by a reference HJB assay) and an elevated risk of invasive pneumococcal disease in young children with HbSC, well described in the literature and further illustrated by the occurrence of a pneumococcal sepsis in a child with low HJB count. Although the HJB assay is (i) an accurate, quantitative technique that correlates well with other reference methods (splenic scintigraphy and manual counts), (ii) consistent with the results expected in splenectomized children and SCA children, and (iii) in line with published data, the HJB count may not accurately reflect the spleen-related risk of invasive pneumococcal disease. While a high count is expected to be associated with an increased risk, we show here that a normal HJB count may not confidently rule out the pneumococcal risk. As a matter of fact, there are no prospectively collected data from the literature to show that HJB counts, pitted cell counts, or even spleen scintigraphy results correlate with the risk of invasive pneumococcal disease. The spleen's ability to clear blood-borne bacteria depends on multiple mechanisms including but not limited to bacterial phagocytosis, mechanical filtration, complement deposition, and antibody response. All of these functions may not be reflected by indirect markers like HJBs or pitted cells. In particular, humoral and cellular immune responses (notably via IgM+ memory B lymphocytes) which play a major role in pneumococcal defense, may not necessarily correlate with HJBs, particularly in children with SCD who experience early microanatomical spleen damage. Altogether, while HJB counts (and especially high counts) provide an overall gauge of the spleen's status, a normal value does not provide a robust negative predictive value for the pneumococcal risk. Taken together, our results show that today's methods for measuring spleen function do not adequately address the risk of pneumococcal infection – the main risk related to spleen impairment in children with SCD. A complementary method for spleen assessment is needed; most probably, it would be based on immunological assays of IgM memory B cells and/or other B cell subsets, in combination with anti-pneumococcal antibody titers. Until specific, reliable immunological markers become available and validated, this work shows that it is reasonable to recommend that anti-pneumococcal prophylaxis be maintained in children with HbSC disease until the age of 5, at least, despite normal HJB counts. None. The authors declare no conflict of interest. The data may be made available upon reasonable request.